The present invention relates to a method and an apparatus for detection of endogenous and synthetic receptor antagonists and receptor modulators, such as e.g. drugs and pharmaceutically active substances. More specifically, the present invention relates to a method and an apparatus based on the use of a miniaturized liquid-based separation technique coupled to a biosensor activated by a receptor agonist and thus giving rise to a measurable response that is affected in a measurable way by the receptor antagonist to be detected.
Biologically active compounds generally lack features enabling sensitive detection thereof by conventional techniques, and their roles in biochemical and physiological processes are therefore often difficult to elucidate.
The detection of biologically active compounds is of particular interest in the pharmaceutical field, e.g. during development of new drugs. Since many native and synthetic substrates constituting commercial drugs act as inhibitors of dysfunctional events in the human body, it is of importance to find systems that enable screening or detection of molecules with that mode of action.
In recent years, there has been an exponential increase in the number of compounds which are interesting for screening. Synthetic libraries from drug companies and natural products have been some of the sources of these compounds. The compounds origin from a broad spectrum of different organisms, such as bacteria, insects, plants and marine organisms. This, together with the introduction of combinatorial libraries for the manufacturing of several thousands of compounds have led to a great demand for new screening techniques which are faster and more selective than the ones used today. Known methods used for drug screening are generally based on pure chemical binding between compounds extracted from, for example, natural products and target molecules, such as receptors, enzymes or nucleic acids. The target molecules can also be included in biological systems, such as living cells, where the merits of chemical recognition and biological amplification are combined.
The use of specific target molecules for the evaluation of a compound""s biological potential is based on the creation of systems of biological relevance for the analyzed compound. Strategies in this field often include expression of cloned cDNA in different cell systems for the production of a functional target molecule in its natural environment.
There are also examples of screening systems which are based on cell effects where the response cannot be traced to a single target molecule.
Several different techniques are presently used for biological screening and characterization of potential drugs, and some examples of these techniques are given below.
Microphysiometry
During the growth of a typical biological cell, carbon-containing nutrients such as glucose are taken up and acidic metabolic products such as lactic acid are released. In microphysiometry these changes in metabolic rate are recorded as changes in the rate of acidification of the medium surrounding the cells (see e.g. Raley-Susman, K. M., et al., J. Neurosci. 12:773, 1992; Baxter, G. T., et al., Biochemistry 31:10950, 1992; Bouvier, C., et al., J. Recept. Res. 13:559, 1993; and McConnell, H. M., et al., Science 257:1906, 1992). Virtually any molecule that affects the cell can be detected by this method. Such molecules include neurotransmitters, growth factors, cytokines and so forth. The microphysiometry is unable to distinguish between different antagonists acting on the same receptor system and can therefore not be used for binary or more complex solutions of such agents. Other drawbacks of this system are the low-level detection, measuring changes in pH is far less selective than measuring responses on the receptor level, and slow recovery rates.
Immunoassays
This group of techniques is based on in vitro procedures for screening of specific antigens (see e.g. Tu, J., et al., Clin. Chem. 44:232, 1998; Pinilla, C., et al., Biomed. Pept. Proteins Nucleic Acids 1:199, 1995; Tawfik, D. S., et al., Proc. Natl. Acad. Sci. USA 90:373, 1993; and Houghten, R. A., et al., Biotechniques 13:412, 1992). Antibodies, often immobilized, are used as targets for antigens. The antigen-antibody interaction is detected by a second antibody, which is labeled by, e.g., a radioactive isotope. The problems with these immuno-based techniques are related to the difficulties in raising specific antibodies for small molecules that are identical or resemble endogenous compounds. Another problem is related to the handling of radioactive substances.
Use of Combinatorial Libraries
Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening (see e.g. Sastry, L., et al., Ciba Found Symp. 159:145, 1991; Huse, W., Ciba Found Symp. 159:91, 1991; Persson, M. A., et al., Proc. Natl. Acad. Sci. USA 88:2432, 1991; Kang, A. S., et al., Proc. Natl. Acad. Sci. USA 88:4363, 1991; Houghten, R. A., et al., Nature 354:84, 1991; Clackson, T., et al, Nature 352:624, 1991; and Ostresh, J. M., et al., Proc. Natl. Acad. Sci. 91:11138, 1991). The libraries are generated by a combination of solution and solid-phase chemistries and are cleaved off the solid-support for screening. When mixtures of compounds are screened, however, the possibility exists that the most active compound will not be identified.
Separation Techniques Coupled to Mass Spectrometry
Separation techniques such as liquid chromatography, gas chromatography and capillary electrophoresis coupled to mass spectrometry or tandem-mass spectrometry create analytical systems available for structure evaluation (see e.g. Hsieh, S., et al., Anal. Chem. 70:1847, 1998; Tretyakova, N. Y., et al., J. Mass. Spectrom. 33:363, 1998; Bonnichsen, R., et al., Zacchia 6:371, 1970; Taylor, G. W., et al., Br. J. Clin. Pharmacol. 42:119, 1996; and McComb, M. E., et al., J. Chromatogr. A 800:1, 1998). Mass spectrometry gives information about the molecular weight of the analyzed molecule. With refined and controlled fragmentation of large molecules it is also possible to extract information about the sequence.
Enzyme Assays Using Proteases
Many proteases have become targets for drug discovery (see e.g. Carroll, C. D., et al., Adv. Exp. Med. Biol. 436:375, 1998; Ferry, G., et al., Mol. Divers. 2:135, 1997; and Jiracek, J., et al., J. Biol. Chem. 270:21701, 1995), from viral proteases required for the generation of active viral proteins to mammalian proteases that process pro-hormones to their active mature forms. Assays have been developed in bacterial systems to screen for compounds that inhibit protease activity. Most of these involve the co-expression of both the protease and a target reporter gene (the gene that encoded the protein which creates a measurable effect) in the same cell. A number of in vitro biochemical assays have also been developed. In most of these cases, a peptide containing the protease cleavage site is labeled at one end using either a radioactive or a fluorescent tag. The other end of the peptide molecule is adhered to a plate or a bead. In the presence of an active protease, the peptide is cleaved and the labeled end is released. The loss of signal from the labeled end of the peptide molecule after washing reflects the activity of the protease and can be easily monitored. For detection of protease inhibitor the grade of maintenance of the signal can instead be measured. These assays are, however, often time consuming since they involve genetic engineering.
A significant limitation of the above mentioned methods is the capacity; the number of compounds that can be rapidly evaluated is extremely low. Alternative methods for high through-put screens are needed.
Another major disadvantage of these known biological screening systems is that they involve extensive multistep purification and isolation of the compounds which are to be tested.
Capillary-based separation methods for identifying bioactive analytes in a mixture have earlier been described in WO 96/10170 (PCT/US95/12444). The methods described herein is however not fully satisfying for detection of antagonists. In the application detection of antagonists is mentioned. However, it seems that the agonist needed for detection of antagonists is then either included in the bathing solution or fed to the cell by a second capillary or tube system. A major drawback with this solution is that the agonist would immediately dissipate from the surface of the cell-based biosensor when the separation process is started. This eliminates the effect of pre-activation which is necessary for accurate biosensor-detection of antagonists.
The disadvantages of the above mentioned methods for drug screening can be eliminated with the method according to the present invention.
The present invention provides methods and apparatus for detection of biologically active analytes separated by liquid-based separation means. The analytes or antagonists to be detected act can act as ligands, which means that they can bind to a specific receptor or receptors. The method employs cell-based biosensors expressing specific receptors to serve as ligand detectors. An important feature of the invention is that the receptor, comprising the functional unit of the detector, is preactivated by constantly including a receptor-specific agonist or modulator into the liquid-based separation means. Analytes, such as antagonists, is detected in that they modulate or inhibit the pre-activated receptor response. The use of a pre-activated or constantly activated biosensor is one of the most important characteristics of the present invention.
Thus, the present invention relates to a method for detection of at least one receptor antagonist and/or at least one receptor modulator, comprising the following steps:
a sample containing the receptor antagonist or modulator is fractionated by use of a liquid-based separation means,
(II) a fraction containing the receptor antagonist or modulator is fed directly to a biosensor (9) which is activated by an appropriate receptor agonist and, as a result of this activation, is generating a measurable response, said agonist being fed to the biosensor through the liquid-based separation means together with the antagonist or modulator, said activation of the biosensor being pulsed by delivery of the receptor agonist to the biosensor for short period of times, said periods being separated by other periods when no agonist is delivered to the biosensor, and
(III) the change of the response resulting from deactivation of the receptor agonist-activated biosensor by the receptor antagonist or modulator is measured. The present invention also relates to an apparatus for detection of a receptor antagonist comprising a capillary electrophoresis separation capillary containing an electrolyte supplemented with an appropriate receptor agonist, the sample inlet part of which is connected to a high-voltage power supply through a buffer vial containing a buffer supplemented with an appropriate receptor agonist and the grounded outlet part of which ends close to a patch-clamped biosensor that is activated by the receptor agonist and deactivated by the fractionated antagonist; the apparatus further comprising a patch clamp electrode and means to record currents detected by the patch clamp electrode.
The method and the apparatus according to the present invention enables a one-step fractionation immediately followed by detection. This is a big advantage compared to known techniques which involves several separation steps.
The present invention also relates to an apparatus and method for better resolution of the separated analytes or antagonists due to a periodic resensitisation of the biosensor by an pulsed flow system.
The methods and the apparatuses according to the invention have a variety of uses. For example, they are well-suited for drug screening since many drugs or pharmaceutically active components are receptor antagonists or receptor modulators, but they can also be used for applications in other areas such as environmental, food, and cosmetic industries.
When the method or the apparatus according to the invention is used for screening of antagonists or drugs they provide a number of advantages over methods according to prior art. Since cell biosensors of the type described herein by themselves are not particularly useful at identifying antagonists or drugs acting as inhibitors in a mixture of antagonists or drugs, it is a great advantage to use the method according to which they are pre-activated by a selective agonist which is incorporated into the running buffer in the liquid-based separation means. An advantage of this system is that the state of the biosensor, that is the degree of receptor activation, is known through this continuous activation, by an agonist. An another significant advantage of the present invention is that long-time exposure of the cell by agonist or antagonist can be avoided by displacement of the cell from the outlet of the liquid-based separation means. This is in contrast to keeping the concentration of the agonist at a high level in the buffer solution in the Petridish, as described in the above mentioned patent application PCT/US95/12444. The fact that the agonist according to the present invention is present in the liquid-based separation means where the separation of the antagonist takes place provides a very important advantage compared to the methods described in WO 96/10170 since this results in a pre-activation of the biosensor enabling accurate detection of antagonists.
The features of the invention will be evident from the following description and the appended claims.